Bacterial Extracellular Vesicle Isolation

Bacterial extracellular vesicles were generated as previously described with some modifications [7 (link)]. Briefly, bacteria were grown up to stationary phase in 120 mL of LB broth prior to being washed twice with 1× PBS and concentrated into 5 mL of PBS. The OD₆₀₀ reading was taken to adjust the bacterial concentration to 10⁸ cell/mL based on previous growth curves. Plating with serial dilution was used to confirm the bacterial count. The bacteria were then inoculated in two 60 mL flasks of fresh LB and grown for 12 h at 37 °C. The contents of each flask were centrifuged at 2000× g for 20 min at 4 °C to pellet out bacterial cells, after which the supernatant was ultracentrifuged at 25,000× g for 20 min at 4 °C. The resulting supernatants were filter sterilized with a 0.22 μm filter into new ultracentrifuge tubes and ultracentrifuged at 150,000× g for 2 h at 4 °C. The vesicle pellets resulting from the two flasks were resuspended in dPBS (Cytivia, Marlborough, MA, USA) and combined prior to a final ultracentrifugation spin at 150,000× g for 2 h at 4 °C before a final resuspension in 500 μL of diluent. Vesicles were stored at 4 °C before being used for quantification assays.